Method for the production of 9-(2-O-acyl-β-D-arabinofuranosyl)adenine compounds

ABSTRACT

9-(2-O-Acyl-β-D-arabinofuranosyl)adenine compounds and their production by enzymatic removal of the 3-O-acyl and 5-O-acyl groups of a 9-(2,3-di-O-acyl-β-D-arabinofuranosyl)adenine compound or a 9-(2,3,5-tri-O-acyl-β-D-arabinofuranosyl)adenine compound. The monoester compounds are useful as antiviral agents. The compounds are water-soluble and lipophilic, thereby being adaptable to a wide variety of pharmaceutical formulations.

This is a division of application Ser. No. 687,271, filed May 17, 1976now U.S. Pat. No. 4,055,718.

SUMMARY AND DETAILED DESCRIPTION

The present invention relates to new organic compounds that are usefulas pharmacological agents and to a method for their production. Moreparticularly, the invention relates to new9-(2-O-acyl-β-D-arabinofuranosyl)-adenine compounds that are representedby the formula ##STR1## where R is a straight or branched chain alkanoylgroup having from 2 to 4 carbon atoms. Examples of alkanoyl groupsrepresented by R are acetyl, propionyl, butyryl, and isobutyryl.

In accordance with the method of the invention,9-(2-O-acyl-β-D-arabinofuranosyl)adenine compounds having formula I areproduced by subjecting to enzymatic removal the 3-O-acyl and 5-O-acylgroups of a 9-(β-D-arabinofuranosyl)-adenine ester compound representedby the formula ##STR2## where R has the same significance and R' ishydrogen or a lower alkanoyl group. The method is carried out bycontacting the ester compound with the enzyme in an aqueous medium untilremoval of the 3-O-acyl and 5-O-acyl groups is achieved. The deacylatedproduct having formula I is isolated from the medium by conventionalrecovery and purification procedures. Enzymes suitable for deacylationare enzymes elaborated by microorganisms of the order Actinomycetales,for example, bacteria of the family Mycobacteriaceae, Streptomycetaceae,Actinomycetaceae, Streptosporangiaceae, and Actinoplanaceae. Alsosuitable are enzymes elaborated by fungi imperfecti. Especially suitableare strains of Bacillus subtilis. Bacillus subtilis is preferred as asource of deacylation enzyme because Bacillus subtilis is widelyavailable and easy to cultivate. The process also can be carried outwith enzymes elaborated by strains of the following exemplary genera andspecies: Streptomyces (mesophil), such as S. viridochromogenes, S.fradiae, S. griseus, S. griseoflavus, S. prasinus; Streptomyces(thermophil), such as S. violaceoruber, Thermoactinomyces vulgaris, S.thermovulgaris; Chainia, such as Actinopycnidium species, Micromonosporaspecies, Nocardia petroleophila, Streptosporangium roseum,Thermopolyspora polyspora, Thermopolyspora glauca, Mycobacteriumtuberculosis var. BCG; Mycobacterium phlei; and Cephalosporium andAspergillus. The preferred acylase, as indicated, is that elaborated bythe microorganism Bacillus subtilis. The acylase itself need not beisolated but a cell paste, mass or mycelium of the desired microorganismcan be used instead.

The aqueous medium for the reaction (or incubation) is maintained at pH6.5 to 7.5, preferably at pH 7.0 to 7.5. The medium is kept attemperatures ranging from 20° to 45° C., preferably 35° to 40° C., untilthe desired deacylation is substantially complete, usually 15 to 24hours. The amount of acylase, or microorganism cell paste or mass usedis not critical. Since the rate of deacylation is dependent on theamount of acylase present, it is convenient and preferable to use excessenzyme, e.g., about equal weights of substrate (starting ester) andmicroorganism cell paste or mass. Following completion of the reaction,the mixture is conveniently freed of solids usually by filtration, andthe desired compound is obtained by conventional means, as indicatedabove.

The 9-(2-O-acyl-β-D-arabinofuranosyl)adenine compounds are new chemicalcompounds that are useful as pharmacological agents, especially asantiviral agents against herpes virus in oral, topical or parenteralform.

Their activity as antiviral agents can be quantitatively measured in anin vitro test by utilizing the plaque reduction technique firstdeveloped by Dulbecco (Proc. Natl. Acad. Sci., Volume 38, pages 747-752)and modified by Hsiung and Melnick (Virology, Volume 1, pages 533-535).In this test, a complete cell monolayer is first grown on a glass testunit. The growth medium is then removed, and the virus is adsorbed onthe cell monolayer for a measured time period. In the absence of anantiviral agent, the virus will destroy well-defined areas of cells,called plaques, that can be seen macroscopically when the vital stain,neutral red, is added to the system. To test the inhibiting effect of agiven compound, the test compound in solution is added to the virus-cellsystem, and the whole is covered with a nutrient agar overlay containingneutral red. After incubation, the plaques are counted, and the numberof plaques produced in the system containing the test compound iscompared with the number produced in the control systems, from whichonly the test compound is omitted. The inhibitory activity of a testcompound is reported as the percentage reduction of the plaque count onthe test units compared with that on the controls.

When tested by this plaque reduction technique, with 4 oz. glass bottlesserving as the test units and H. Ep. No. 2 cells making up the cellmonolayer, compounds of the invention, at a concentration of about 15 to60 micrograms/ml. in Hank's Balanced Salt Solution (pH 7-8), typicallywere found to give substantially complete plaque reduction againstherpes simplex.

The ester compounds of the invention structurally resemble9-(β-D-arabinofuranosyl)adenine, which is known to be an antiviral agentthat is active against herpes virus. The latter compound has beenreported to be more active in vitro against herpes virus than its5'-benzoyl ester whereas its 5'-palmitate ester was inactive in the sametest (Renis et al., J. Med. Chem., 16, 754); the compound has also beenreported (Repta et al., J. Pharm. Sci., 64, 392) to be poorly soluble inwater (and subject to enzymatic deamination in the correspondingbiologically inactive hypoxanthine) and its 5'-formate ester, relativelywater-soluble, to be unstable in aqueous solution. Other relativelypoorly water-soluble esters of 9-(β-D-arabinofuranosyl)adenine are thetriesters described in U.S. Pat. No. 3,651,045. It is thereforesurprising that the compounds of the invention, unlike the prior artcompounds, exhibit good antiviral activity, are resistant to enzymaticdeamination, and are adaptable to aqueous and non-aqueous pharmaceuticalformulation, being readily soluble in water and/or being lipophilic.Preferred compounds of the invention in this regard are9-(2-O-acetyl-β-D-arabinofuranosyl)adenine and9-(2-O-propionyl-β-D-arabinofuranosyl)adenine.

The invention is illustrated by the following examples.

EXAMPLE 1

To a stirred suspension of 500 mg. of9-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)adenine (U.S. Pat. No.3,651,045, supra) in 50 ml. of a 0.1 M pH 7 phosphate buffer is added500 mg. of Bacillus subtilis ATCC 6633 cell paste or lyophilizate (U.S.Pat. No. 3,304,236, Example 9) and the mixture is stirred for 22 hoursat 35°-40° C., with periodic addition of saturated aqueous sodiumbicarbonate to maintain the pH at 7.0 to 7.5. The incubated mixture isthen poured into 150 ml. of methanol and the resulting mixture isfiltered through diatomaceous earth and the filtrate evaporated atreduced pressure. The residue is passed through a 1×30 cm. column of drysilica gel, and the column is eluted sequentially with 5:95, 10:90 and20:90 (v/w) methanol-chloroform. The eluate is collected in 10-ml.fractions and those that contain the desired product, as established bythin layer chromatography, are combined and evaporated at reducedpressure to give the desired product9-(2-O-acetyl-β-D-arabinofuranosyl)adenine; λ_(max) ^(CH).sbsp.3^(OH)=259 nm. The structure is confirmed by nmr spectra.

By substituting 500 mg. of9-(2,3-di-O-acetyl-β-D-arabinofuranosyl)adenine for the9-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)adenine in the aboveprocedure, the same end product is obtained.

EXAMPLE 2

By substituting 500 mg. of either9-(2,3,5-tri-O-propionyl-β-D-arabinofuranosyl)adenine or9-(2,3-di-O-propionyl-β-D-arabinofuranosyl)adenine for the9-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)adenine in Example 1, theproduct obtained is 9-(2-O-propionyl-β-D-arabinofuranosyl)-adenine; m.p.206.5°-207.5° C. after crystallization from ethanol, λ_(max)^(CH).sbsp.3^(OH) =259 nm (ε=14,600), partition coefficient, 1.55(pentanol/water).

EXAMPLE 3

By substituting 500 mg. of either9-(2,3,5-tri-O-isobutyryl-β-D-arabinofuranosyl)adenine or9-(2,3-di-O-isobutyryl-β-D-arabinofuranosyl)adenine for the9-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)adenine in Example 1, theproduct obtained is 9-(2-O-isobutyryl-β-D-arabinofuranosyl)adenine.

Preparation of Diacyl Ester Starting Materials

The 9-(2,3-di-O-acyl-β-D-arabinofuranosyl)-adenine starting materialsspecified above are new compounds. These compounds can be prepared fromknown materials by the following procedure.

(a) To a well-stirred suspension of 26.7 g. of9-β-D-arabinofuranosyladenine in 500 ml. of dry dimethylformamide,containing 16.3 g. of imidazole, is added 18.1 g. oftert-butylchlorodimethylsilane. The mixture is stirred, with protectionfrom moisture, for 20 hours at room temperature, then evaporated atreduced pressure at 50°-60° C. The residue is dissolved in 300 ml. ofethyl acetate and the solution is washed with water, dried andevaporated at reduced pressure. The residual syrup is dissolved in 240ml. of hot chloroform; the solution is diluted to cloudiness with hexaneand cooled to crystalline9-[5-O-(tert-butyldimethysilyl)-β-D-arabinofuranosyl]adenine, which iscollected by filtration, washed with hexane and dried at 80° C. atreduced pressure; m.p. 157°-158° C., [α]_(D) ²³ =+4.1°, λ_(max)^(CH).sbsp.3^(OH) =259 nm (ε=15,000).

(b) To a well-stirred solution of 15.4 g. of9-[5-O-(tert-butyldimethylsilyl)-β-D-arabinofuranosyl]-adenine in 200ml. of dry pyridine is added 9.44 ml. of acetic anhydride. The solutionis stirred at room temperature for 16 hours, treated with 100 g. ofchipped ice and stirred one additional hour. The resulting solution isevaporated at reduced pressure at 45° C. and the residue is dissolved in250 ml. of chloroform. The chloroform solution is washed with aqueoussodium bicarbonate and with water, and is dried and evaporated. Theresidual product,9-[2,3-di-O-acetyl-5-O-(tert-butyldimethylsilyl)-β-D-arabinofuranosyl]adenine,is suitable for use as a starting material for the procedure ofparagraph (c) without further purification.

(c) The product of (b) is dissolved in 300 ml. of tetrahydrofuran, thesolution is treated with 2.3 ml. of glacial acetic acid and 31.3 g. oftetrabutylammonium fluoride and allowed to stand at room temperature for2 hours. The solution is then passed over a 5×10 cm. column of drysilica gel. The column is eluted with one liter of tetrahydrofuran andthe eluate is evaporated at reduced pressure to give the product9-(2,3-di-O-acetyl-β-D-arabinofuranosyl)adenine; m.p. 138°-139° C. aftercrystallization from acetone, [α]_(D) ²³ =-4.1° (c=1% in methanol),λ_(max) ^(CH).sbsp.3^(OH) =259 nm (ε=15,000).

(d) From 15.0 g of9-[5-O-(tert-butyldimethylsilyl)-β-D-arabinofuranosyl]adenine and 11.1ml. of propionic anhydride in 100 ml. of dry pyridine, following theprocedure of (b), there is obtained9-[5-O-(tert-butyldimethylsilyl)-2,3-di-O-propionyl-β-D-arabinofuranosyl]adenine,which, on reaction with 31.3 g. of tetrabutylammonium fluoride in 200ml. of tetrahydrofuran and 2.3 ml. of glacial acetic acid, following theprocedure of (c), gives9-(2,3-di-O-propionyl-β-D-arabinofuranosyl)adenine; m.p. 172°-173° C.after crystallization from acetone, [α]_(D) ²³ =-4.1° (c=1% inmethanol), λ_(max) ^(CH).sbsp.3^(OH) =259 nm (ε=15,000). From 1.79 g. of9-[5-O-(tert-butyldimethylsilyl)-β-D-arabinofuranosyl]adenine and 2.34ml. of isobutyryl chloride in 50 ml. of dry pyridine, following theprocedure of (b), there is obtained9-[5-O-tert-butyldimethylsilyl)-2,3-di-O-isobutyryl-β-D-arabinofuranosyl]adenine,which, on reaction with 3.7 g. of tetrabutylammonium fluoride in 100 ml.of tetrahydrofuran and 0.5 ml. of glacial acetic acid, following theprocedure of c), gives9-(2,3-di-O-isobutyryl-β-D-arabinofuranosyl)adenine; m.p. 207°-208° C.after crystallization from acetone, λ_(max) ^(CH).sbsp.3^(OH) =259 nm(ε=15,000).

I claim:
 1. A method for the production of a9-(2-O-acyl-beta-D-arabinofuranosyl)adenine compound having the formula##STR3## wherein R is a straight or branched chain alkanoyl group havingfrom 2 to 4 carbon atoms which comprises contacting a9-(beta-D-arabinofuranosyl)adenine ester compound represented by theformula ##STR4## where R has the same significance and R' is hydrogen ora lower alkanoyl group with an acylase capable of deacylating the 3 and5-O-acyl groups; said contacting being carried out at 20°-45° C. at a pHof 6.5-7.5, and recovering the product.
 2. Method according to claim 1where the acyl removal is carried out at temperatures ranging from 35°to 40° C. in an aqueous medium at pH 7 to 7.5 using approximately equalweights of the ester compound and enzyme elaborating microorganism cellmass.
 3. Method according to claim 2 where the enzyme elaboratingmicroorganism is a strain of Bacillus subtilis.
 4. Method according toclaim 1 where the ester compound is a 2-O-acetyl ester compound. 5.Method according to claim 1 where the ester compound is a 2-O-propionylester compound.